JPS62501327A - Method and composition for detecting familial hypercholesterolemia - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Method and composition for detecting familial hypercholesterolemia The present invention can reduce hypercholesterolemia, atherosclerosis and ultimately heart disease. The present invention relates to methods and compositions useful in diagnosing genetic disease predisposition. Change Specifically, the present invention provides a method for treating individuals suspected of having familial hypercholesterolemia Useful for determining the presence of a mutant low-density riboprotein (LDL) receptor gene It is associated with recombinant DNA molecules that serve as useful probes.

Half of all deaths in the United States are attributable to atherosclerosis.

Atherosclerosis is a condition in which cholesterol builds up on the walls of your arteries and eventually forms blood clots. Forms bulky plaques that block blood flow and block arteries until It is a disease that causes heart attacks. Cholesterol in atherosclerotic plaques are particles called low-density riboproteins (LDLs) that circulate in the bloodstream. is induced from The more LDL there is in the blood, the more atherosclerosis develops. Get sick quickly.

Epidemiological data show that half of the people living in Western industrialized countries, including the United States, The above is a level of LD that puts them at a very high risk of developing atherosclerosis. A surprising fact has been discovered that it has an L circulation level. If such a concentration is It is considered “normal” because it is so common, but clearly it is not. Not always. People are increasingly prone to atherosclerosis and heart attacks be.

Regarding why LDL levels are dangerously high for many Americans Some answers come from research into a special protein called the LDL receptor. There is. This receptor protrudes from the surface of animal cells. The receptor is LDL particle extract the particles from the liquid that binds and bathes the cells. LDL is taken into cells It is destroyed and produces cholesterol to meet the needs of each cell. Corres in cells When supplying terol, receptors are important in preventing atherosclerosis. It serves a second physiological function. That is, the receptor removes LDL from the blood stream.

The number of receptors present on the surface of a cell depends on the cell's demand for cholesterol. Change. With less demand, excess cholesterol accumulates and cells have fewer It produces a large number of receptors and takes up LDL at a low rate. This allows the cells to absorb excess It protects you from cholesterol, but it comes at a high cost. Is the decrease in the number of receptors related to the circulatory system? This decreases the rate at which LDL is removed from the body, increases the blood concentration of LDL, and increases the risk of atherosclerosis. Sclerosis is accelerated.

High LDL concentrations in many Americans reduce production of LDL receptors It has been said that the cause is a combination of factors. The central role of receptors has been recognized This led to the treatment of aggressive genetic forms of atherosclerosis and the general shed light on the controversy regarding the role of dietary restriction on atherosclerosis in the global population. I decided to throw it.

LDL consists of approximately 150 oily liquids each linked to a long-chain fatty acid through an ester bond. It is a large spherical particle consisting of 0 molecules of fatty alcohol cholesterol. Coles This core of terol esters contains phospholipids and unesterified cholesterol molecules. is surrounded by layers of Phospholipids are arranged so that their hydrophilic head is on the outside. are arranged in a sequence that allows LDL to dissolve in the blood or intercellular fluid. . Buried in this hydrophilic coating is a protein named apoprotein B-100. is a large protein molecule.

Recognized and bound by LDL receptor, glycoprotein (protein to which sugar chains are attached) It is apoprotein B-100. Receptors are spread throughout the thickness of the cell's plasma membrane. It carries a binding site that protrudes from the cell surface. The binding is LDL month 0- Occurs when present at concentrations below 9M. That is, the receptor contains more than a billion water molecules. One LDL particle can be picked up from. The receptor is apoprotein B-10 0 or a related protein named apoprotein E. Join.

In 1976, it was discovered that the cell membrane (the inner surface of the underlying membrane contains the protein clathrin). craters known as coated pits (because they are coated by in) LDL receptors may cluster in the special region that they are trying to form. It's been found. Within minutes of formation, the pits become internalized and cut from the surface. Separated to form a membranous marginal capsule called the capsularis. LDL bound to receptor is transported into the cell. Applicable to this method taken by covered pits and sacs Receptor-mediated endocytosis, a term used to describe It is recognized as a general mechanism for uptake of many large molecules that have specific receptors. .

Eventually, LDL is separated from the receptor (receptor is recycled to the cell surface) and LDL is transported to lysosomes, which are sacs filled with digestive enzymes. enzyme one destroys the covering of LDL and exposes the cholesterol ester core. other enzymes cleaves the fatty acid tail of cholesterol ester to produce non-esterified cholesterol. This releases the molecules, which leave the lysosomes. Whole cells have newly synthesized surfaces Incorporate cholesterol into membranes. In certain specialized cells, LDL is derived from Cholesterol has another role. In the adrenal glands and ovaries, cholesterol is converted into the steroid hormones cortisol and estradiol, respectively. In the liver, cholesterol is used to produce bile acids, which have a digestive effect in the intestines. It is converted to .

The central role of the LDL receptor in atherosclerosis was first recognized in 1974. The lack of the receptor causes a severe condition called familial hypercholesterolemia (FH). It has been shown that it causes many diseases. Long before that, in 1939, Noru Oslo Community Hospital Carl Mullar of o5pital was diagnosed with this disease. Qi is a congenital condition that causes high cholesterol levels in the blood and heart attacks in young people. It is considered a metabolic disorder and is transmitted as a dominant predisposition determined by a single gene. I realized that. In the 1960s, two forms of the disease were identified: heterozygous and and more severe homozygosity were reported. A transzygote carries one mutant gene. It is inherited and quite common. In many races, about 1 in 500 people be. Their plasma LDL levels are twice the normal level (even before birth). By the age of 35, he began having heart attacks. People over 60 who have had a heart attack Of these, 1 in 20 people have heterozygous FH.

If two FH heterozygotes marry (1 in 250,000 couples), 1 in 4 children will be born. A person inherits two copies of the mutant gene (one from each parent). There is a possibility that it will be transmitted. Such PH homozygotes (approximately 1 in 1 million people) Children with circulating LDL levels six times higher than normal are at risk for heart attacks starting at the age of two. Yes, it is almost inevitable that a heart attack will occur before the age of 20. like this Children have no risk factors for atherosclerosis other than high LDL levels. It is noteworthy. They have normal blood pressure, do not smoke, and have high blood levels of glucose. Has no degree. Homozygous FH is a lively experiment of nature. That's obviously , showing a causal relationship between high circulating LDL levels and atherosclerosis.

Heterozygotes with familial hypercholesterolemia have blood plasma from the umbilical cord. contains 2 to 3 times higher concentrations of LDL-cholesterol, so it is often Can be suspected at birth. High levels of plasma LDL persist throughout life, but are typical Symptoms do not appear until the person's 30s or 40s. The most important therapeutic requirement is early This is accelerated coronary atherosclerosis. Myocardial infarction occurred in a male patient in his 30s. Initially, the incidence peaks in people in their 40s and 50s. At the age of 60, approximately 85% of the heart Have experienced an infarction. In women, the incidence of myocardial infarction also increases, but Their average age is 10 years later than that of men. Heterozygotes for familial hypercholesterolemia are , accounting for approximately 5% of myocardial infarction patients.

Key xanthomas are the second major clinical manifestation of the heterozygous state. Xanthomas are Achilles Nodular swelling involving the muscles and other muscles around the knees, elbows, and backs of the hands. So This is a tissue macrophage in which LDL-derived cholesterol esters exist in the interstitial spaces. It is formed by depositing on the stage. Macrophages swell with lipid droplets and form joal cells. Cholesterol causes eyelid softness It also deposits in the tissue, causing xanthomas, and a lipoid ring on the cornea. company yellow Xanthomas are an essential sign of familial hypercholesterolemia, but xanthomas and lipomas Ido ring cornea is not specific. The latter two abnormalities are due to normal plasmid lipid levels. It also occurs in many adults. Incidence of xanthomas in familial hypercholesterolemia increases with age. Eventually, approximately 75% of heterozygous patients will show this sign. vinegar.

Homozygous patients exhibit significantly higher plasma LDL levels from birth. Germany A particular type of planar skin xanthomas is often present at birth and in the last six years of life. Always appear. This kind of skin xanthomas affects skin areas such as the knees, elbows, and upper buttocks. It is a raised, yellow, plaque-like lesion that occurs on the traumatized area of the skin. Xanthoma is is almost always present in the interdigital tissue, especially between the thumb and index finger. key xanthomas, Poid ring cornea and xanthomas are also specific. Coronary atherosclerosis Symptoms often begin before the age of 10 in zygotes, and myocardial infarction can occur as early as 18 years. reported in months. In addition to coronary atherosclerosis, cholesterol is often , deposits on the aortic valve and causes symptoms of aortic stenosis. Typically, homozygous patients have 3 The patient died before the age of 0 due to complications of myocardial infarction.

In many populations, 1 in 500 people will have a heterozygous family with disrupted gene function. Mutation in LDL receptor gene that causes familial hypercholesterolemia syndrome have. Many of these mutated genes are unable to produce detectable receptors. I can't. Other mutated genes produce only a few receptors. Sara Other mutated genes have essentially normal numbers but do not bind LDL properly. produce defective receptors.

In other patients, abnormally long LDL receptor proteins are produced. for gel electrophoresis The normal precursor of the receptor exhibits an apparent molecular weight of about 120,000 as measured by However, the abnormally shaped protein encoded by the mutant gene is 100,000,+ The apparent values of 35,000 and 170,000 were found to shift to the molecular weight position. . These mutations found in the LDL receptor protein may be responsible for LDL receptor production. may be the result of a deletion or insertion mutation in the responsible gene. described earlier All of the mutations appear to be in or near the gene for the LDL receptor. be loyal to

Therefore, there is no way to directly identify individuals with a mutant LDL receptor gene. If there are steps, individuals with a genetic predisposition to atherosclerosis and heart attacks Your ability to distinguish between people will be greatly improved. If complementary to the receptor gene Once DNA (cDNA) is discovered, such mutations can be confirmed. It will be possible.

Figure 1 shows the cDNA cloning procedure used to clone the human LDL receptor. It is a schematic representation of the law. Human λh1 corresponds to the 3′ end of the LDL receptor gene. This is a partial genome clone. Recombinant plasmids PIO1 and p203 are human Contains a partial CDNA that is the complement of the human LDL receptor mRNA. plus Mid-pLDLR-2 is a fusion construct of plol and p203, Contains almost full-length cDNA for L receptor mRNA. in parentheses The numbers inside indicate the length of the DNA insert into the given clone.

Figure 2 contains nearly complete length cDNA for the human L'DL receptor gene. Figure 2 is a structural representation of the recombinant plasmid pLDLR-2. Decoding area (hatched area) contains nucleotides 1-2580. This fusion plasmid is p203 (nucleotides 1-323) and plol (nucleotides 324-5144) cDNA insert was constructed by joining the HglAl site overlappingly. It is something. The filled regions in pLDLR-2 indicate the origin of replication, 16S and Contains 19S donor and acceptor snip sites and polyadenylation signals The region in the cloning vector containing the SV40 sequence is shown.

Figure 3 shows the nucleotide sequence and cDNA corresponding to human LDL receptor mRNA. and the predicted amino acid sequence of the receptor protein. Nucleotides start from 5° Numbered on the right in the direction to 3', nucleotide l is the initiator methion This is the A of the ATG codon that encodes Nin. Negative numbers indicate 5° untranslated regions. vinegar. Amino acids are numbered below the sequence. Residue l is N in the mature protein H is an alanine found at the end. Negative numbers indicate cleaved signal sequences.

Signal sequences (21 residues) and membrane span sequences (22 residues) are underlined with one solid line. It is shown.

A site to which an N-linked carbohydrate can be attached (Asn-X-9er or A 5n-X-Thr) is shown with a double solid underline. The cysteine residue is , circled. o - Serines and threads to which binding carbohydrates can bind One range of 48 residues that is rich in onine is indicated by dotted underlining. cDNA The Alu sequence in the 3' untranslated region is boxed. Direct reaction associated with the first Alu arrangement Reductions are indicated by dashed arrows above the array. 3 possible points in the untranslated region The rearenylation signal is indicated by overlining and underlining.

Figure 4 shows DNA probes obtained from different regions of the LDL receptor protein cDNA. Normal people and people with familial hypercholesterolemia (named PH274) The results of analysis of the Xba upper restriction digest of genomic DNA of . Figure 4A shows the translation region Humans shown with AUG and UGA without indicating the beginning and end of, respectively. 1 is a diagram of mRNA for the LDL receptor. LDL receptor gene The size and size of the 3'P-labeled cDNA probes used to create the map. The positions are indicated by filled bars and numbered 1-9. Figure 4B shows the Normal (norma) after crossing with Lobe 1 (left) or Probe 8 (right) +) or mutant (Fl-1274) genomic DNA. (Southern blot). Xbal-Restrictions Fragments are indicated A-D to the right of each plot. molecular size standard were generated by HindlIl cleavage of bacteriophage DNA, and each shown on the left side of the page. TsumDN in probes 2-9 in normal subjects and PH274 A Southern plot cross of A is shown. By culturing fibroblasts obtained from the indicated subjects. The isolated genomic DNA (5 μg) was digested with Xbal, subjected to electrophoresis, The product was transferred to nitrocellulose and cleaved by interaction with 3Np-probe 1.

Figure 6 shows the normal LDL receptor gene and three deletion-containing genes from PH274. ° Comparison of terminal restriction maps. The lower scale is the length unit of genomic DNA: kilobase). The composition of the normal LDL receptor gene is shown at the top of the figure. . Exons (informational sequences) are indicated by filled segments and uppercase letters. . Intervening sequences (IVS) are indicated with unfilled segments and lowercase letters. . Alu repeats in C of IVs and F of exon are shown. PH27 Restriction enzyme recognition sites used to determine gene deletions in 4 are indicated.

The present invention corresponds to the mRNA sequence of the human low-density riboprotein (LDL) receptor gene. Discloses techniques suitable for constructing recombinant DNA transfer vectors containing cDNA sequences for Ru. In addition, the present invention provides the human LDL receptor gene or the mRNA transcription of the gene. Recombinant D containing DNA inserts that are complements to various parts of either A NA transfer vector is disclosed.

The recombinant DNA transfer vector disclosed by the present invention is useful for carrying out the present invention. It is not necessary to necessarily contain the entire human LDL receptor gene. Similarly, The recombinant transfer vector contains a DNA flag that is the complement of the entire mRNA transcript of the gene. There is no need to include ment. The recombinant transfer vector contains the human LDL receptor gene or is a smaller subfragment that is the complement of either of the mRNAs of the gene? It may be configured as follows.

In fact, the use of different subfragments of genes as probes is This will be necessary to elucidate specific mutations in FH patients. everything you need is sufficient for these fragments to form stable duplexes or hybrids. It is important to have sufficient length. Such fragments allow stable duplex formation. This is why they are said to be ``hybridable.'' Generally at least 14 nucleos DNA fragments with tide are stable duplexes (i.e., tetradecamers) can be formed.

People with familial hypercholesterolemia (PH) can use this invention to Diagnosis is made by determining the presence of a mutation in the gene that codes for the condition. Ru. The DNA isolated from the recombinant cDNA clone was isolated from a family member with a deletion in the gene. It has been used to diagnose patients with hypercholesterolemia. cDNA or its fragments contain other mutations containing single nucleotide exchanges (point mutations). Of course, it is useful for diagnosing mutations.

Generally, the method involves disrupting DNA obtained from cells of a person suspected of carrying the mutation; The DNA fragment is then characterized by certain physicochemical properties of the DNA, e.g. It consists of separating the fragments into patterns according to their molecular weight or size. separation The extracted DNA is then extracted from the component corresponding to the LDL receptor gene. The cross-probing is performed with labeled LDL receptor DNA to identify the target. So From this, the pattern of LDL receptor gene fragments in suspected patients was determined. By comparing with similar fragment patterns obtained from normal people, it is possible to identify patients. A person can determine whether they exhibit the mutation or not. If someone seems to be a patient The LDL receptor gene fragment obtained from If it shows, it means that a genetic mutation has been detected.

One method that has proven particularly useful for cleaving DNA is restriction enzyme digestion. This is a method that uses However, other methods, including, for example, chemical cleavage of DNA , the method reproducibly cleaves genomic DNA into the same isolated fragments. If so, you can use it.

Split DNA can be separated into recognizable patterns using various methods. The most useful methods are those that utilize different sizes of isolated DNA. It is. For example, DNA fragments can be isolated by velocity sedimentation using a density gradient. Separate according to molecular weight or by molecular size using exclusion chromatography can be done. However, the preferred method for the purposes of the present invention is to DNA is separated by electrophoresis using a polyacrylamide gel matrix or a polyacrylamide gel matrix. It is to separate the fragments.

Cloned LDL receptor DNA is conveniently obtained by hybridization and autoradiography. Afterwards, the corresponding genomic LDL receptor DNA fragment can be easily visualized. It can be labeled with a radioactive nucleotide that can be used. For example, heavy isotopes Although other labeling methods are possible, including would actually be a hassle.

In addition to the use of cloned DNA fragments, portions of the cDNA disclosed in this invention may also be used. In principle, diagnosis can also be performed using chemically synthesized oligonucleotides corresponding to I can do it. Genomic DNA obtained from a person with a single base substitution in the LDL receptor gene A interacts weakly with such oligonucleotides compared to DNA from a normal person. It's complicated. Such weakened crosses therefore result in heterozygous and homozygous Both are diagnostic methods for many patients with FH.

Low-density riboprotein (LDL) receptors regulate cholesterol in humans and animals. It is a cell surface protein that plays a central role in the metabolism of alcohol.

During the process of endocytosis, LDL receptors bind serum cholesterol. It is responsible for making it available for cell metabolism. This is the receptor/co Enabled by the uptake of the Lsterol complex, and then the incorporated complex Cholesterol is liberated through catabolism.

Free cholesterol influences the rate of LDL receptor synthesis through a feedback mechanism. Adjust. in certain steroid biosynthetic tissues such as the adrenal cortex and the ovarian corpus luteum. The increased cholesterol requirement in the human body is met by an increased number of LDL receptors. . The first notable property of the LDL receptor influences its structure and function. Familial hypercholesterolemia, one of the most commonly mutated human genetic diseases This is because it is causing blood cellemia.

Recombinant DNA technology can be used to detect the presence of mutations in individuals thought to have FH. We offer one way to do so. Purified human LDL receptor gene or its hybrid By using probes made up of potentially heterogeneous subfragments, Abnormalities present in the LDL receptor gene can then be discovered, and the individual then , F) may receive other types of treatment directed at the symptoms of (i.e. If a PH patient is found by the method disclosed by the present invention, the patient should lowering a patient's circulating cholesterol levels, including physical limitations, medications, and surgery. It can be treated as a target for treatment. In addition, LDL in PH patients Knowledge of the genetic structure of the receptor gene, including somatic substitutions or modifications, This will ultimately lead to breakthrough treatments for the disease.

The first step in estimating and confirming the genetic abnormalities present in FH patients is to Genetic engineering techniques can be used to study normal and abnormal LDL receptor genes. development of appropriate probes that can be used. Studying the structure of the human LDL receptor gene The most ideal genetic probe for this purpose would be the cloned human LDL receptor gene or or cDNA prepared for receptor mRNA. However, this problem In order to directly address the problem, it is said that it is necessary to separate the probe from a certain human source material. There are difficulties. This raw material preferably contains the receptor and its mRNA. Probably the adrenal glands or ovaries, which are present in large numbers. This technique is suitable for a suitable human with sufficient m. It is somewhat impractical in that tissue is difficult to obtain. The present invention L receptor cDNA was cloned from raw materials, and cloned female LDL receptor cDNA The idea was to use A to separate human gene parts from a gene library. Describe the methods to avoid this. The portion of the human gene is then converted into a nearly full-length human c Used to isolate DNA clones. This technique can be used as a hybridization probe. The sequence used correctly probes the human LD receptor gene. c.D.N.A. is not clear until it is cloned and sequenced. That's something unexpected. Looking back, we can see that the difference between cow and human genes. Homology is sufficient to enable the cloning approach detailed herein. there were.

Isolation of human recombinant DNA clones carrying a copy of human LDL receptor mRNA has facilitated the development of assays that can detect LDL receptor gene mutations. did. To illustrate the usefulness of this analysis, a sample of patients with familial hypercholesterolemia A case study was conducted on a patient (hereinafter referred to as PH274). write it here case studies provide detailed information about the makeup of a patient's specific genetic defect. However, the present invention contemplates that such measurements are necessary as a diagnostic method. It's not. However, this content does not reflect those made available by the present invention. It is included here to demonstrate the power of genetic technology. Furthermore, these technologies , provides a means to diagnose many other defects in the LDL receptor gene.

That is, the present invention discovers the existence of a genetic defect in the LDL receptor gene. Not only that, but also a method that can enumerate specific genetic defects in great detail. I will provide a.

The inventors believe that these techniques address the presence of genetic defects in the LDL receptor gene. We believe this will provide a way to screen the population as a whole.

Once a patient is detected using the screening methods detailed here, the next step is to detect sudden changes. Specific abnormalities exhibited by different LDL receptor genes can be studied in more detail. Wear. Accordingly, Applicant has disclosed that the present invention relates to a significant and common human genetic disorder. It also provides a better understanding of the genetic phenomena that cause familial hypercholesterolemia. I think that.

Example 1 Cloning of bovine LDL receptor gene cDNA Cloning of bovine LDL receptor gene cDNA loan (hereinafter referred to as pLDLR-1).

) were isolated by polysome immunopurification and oligonucleotide hybridization. general , the method progressed through five stages. These steps are: (1) medial receptor protein; (2) production of polyclonal antibodies capable of reacting with bovine LDL receptor protein; (3) Specific immunoprecipitation of bovine polysomes actively involved in the translation of Namamugi mRNA , followed by enrichment of receptor mRNA isolated from precipitated polysomes, (4) enrichment Preparation of cDNA clone bank from mRNA, (5) Bovine LDL receptor cDNA cDNA clone bank screening to isolate representative clones with inserts. Leaning. These stages are detailed below.

Separation of raw barley and production of polyclonal antibodies Schneide r) et al., Journal of Biological Chemistry (J, Biol, Chern, ), 257: 2664-2673 (1982). Then, homogeneous LDL receptor protein was isolated from the semi-open kidney cortex. Half-open LDL reception Polyclonal antibodies against T. Cell, 30: 715-724 (1 Staphylococcal protein A-purified on Sepharose as described in 982) did. This antibody and the corresponding non-immune rabbit IgG can be used in polysomes or Crude ribonuclease as shown by its inability to change the sedimentation behavior of sugar gradients. It did not contain any contaminants.

Polysome immunoprecipitation of bovine LDL receptor mRNA mRNA for LDL receptor Polysomes enriched with Immerse living tissue in liquid nitrogen, Killed it within 5 minutes. Liquidize the adrenal glands using a Warning blender. It was ground in nitrogen and stored at -70°C until polysome isolation.

10 g of crushed adrenal glands was added to a Brickman Polytron. 25mM Tris-HCl (pH 7.5, 25mM NaCg) M, MgC (!t5mM, Triton)-1002% (v/ v), heparin 0.3 mg/m (!, trichodermin n) 1 ug/mQ, phenylmethylsulfonyl fluoride in 0 μg/m It was homogenized with Balmiter (P almiter), biochemistry (B iochemistry), 13: 3606-3615 (1974). Polysomes were separated from the homogenate by MgC (b) precipitation and incubated at -70 °C. It was stored in Polysome 25A per gram of adrenal powder. I got credit.

A2B on a linear gradient of sucrose. Approximately 70% of the material precipitated as polysomes . The remaining absorbent was present in 80S monosomes. Polysome (100oAf @O units) was clarified by centrifugation at 20,000 xg for 10 minutes, and added with 25mM Tris salt. Acid (pH 7.5, NaCQl 50mM, MgC (2°C 5mM).

Nonidet P-400, 1%, heparin 0, 2 mg/ mQ, ) 15A2s in a buffer containing lycodermin lμg/mQ). /111 12 and stirred with 6.25 mg of anti-receptor 1gG or non-immune IgG. The cells were cultured for 1 hour at 4°C.

Next, the polysome/antibody slurry was diluted with the above dilution buffer at 8 to 10 mQ/hour. A column of protein A-Sepharose (0.7 x 13 cm) equilibrated at a flow rate of ) twice. The column was washed overnight with 120 mC dilution buffer. Combined port Lysomes were treated with 25n+M Tris-HCl (pH 7, 5, 20mM EDTA) in 20m 12 at maximum flow rate. The eluted fractions were heated at 65°C for 5 min and the 0.5M Added NaC (! and 0.2% NaDodSOa, cooled to 24 °C, 10 mM Oligo(dT)-cell equilibrated with Tris-HCl (pH 7, 5,0,5M NaC) Passed through a loin column (0.8 x 2.3 cm).

Wash the column with this buffer 20+nf2 and remove poly(A)'' RNA (IOmM). Elution was performed with 5me of lithium hydrochloric acid (pH 7,5). East carrier tRNA (50 μg) was added and the RNA was precipitated twice with Na0Ac and ethanol. child The immunopurified poly(A)'' RNA was rehydrated in 20 μg of water and incubated at -70°C. Stored.

Immuno-selected poly(A)” RNA was added to the reticular array in response to the presence of LDL receptor mRNA. Measured by in vitro translation in a red blood cell lysate system. The lysate type is below explain.

Bo1baA)"mRNA was incubated with 2.5mM CH3T (goH) at 4°C. incubate for 0 min, then incubate with Pelham and Jacman. kson), European Journal of Biochemistry (Eur, J.

Biochem, ), 67: 247-256 (1970). 80mM KOAcS, 1mM Mg(OAc)t, 19 amino acids ( 16 μM each (excluding methionine) and 0.2 iCi of [35s]methionine (I In rabbit reticulocyte lysate supplemented with C1 = 3゜7 x 1010Bq) Translated by .

The final concentrations of CH and HgOH in the translation reaction were 0.3 mM. translation The translated products were subjected to electrophoresis using a NaDodSO4/polyacrylamide 7% gel. Analyzed by.

As determined by NaDodSO+ gel electrophoresis and fluorography of the synthesized product. The total adrenal poly(A) RNA was directed toward the synthesis of many proteins. Poly(A)” RNA derived from purified polysomes has a certain protein band. Several species and one obvious addition: towards the synthesis of proteins transferred with Mr of about 120,000. I was being kicked. ° This protein was selected from polysomes by non-immune IgG After translating poly(A)” RNA, it could not be proven. Hamsters and rabbits Research into the biosynthesis of 'LDL receptors from Initially created as Mr precursor (where Mr represents the molecular weight in Daltons) This undergoes a series of post-translational glycosylation during transport to the cell surface, resulting in , resulting in a mature protein with an apparent Mr of 160,000. Therefore, the immunoselected poly( A) “The size of enriched proteins seen after translation of RNA is similar to that of the LDL receptor precursor. It matched the size.

Preparation and Screening of a Bovine cDNA Clone Bank a) and Berg, Molecular and Cellular Biolo Zee (Mo1. Ce11. Biol, ), 2: I 61-170 (1982) in polysomes. derived from unit To construct a cDNA library from poly(A)" RNA, immunoselected poly(A)" A) = RNA was used. Life science (L1reS science) Yeast obtained from Ohamibi P-L Biochemicals For cloning reactions using primer, 1.4 μg of dT-tiled vector primer and 0.52 pmol of dG-tailed linker were used.

Maniatis et al., Molecular Cloning cular Cloning) [Cold Springs + Harbor Live Larry, Cold Springs + Harbour. borLibrary, Co1d Spring Harbor), Newyo By the Ca CQ t-shock method described on page 250, Converting Escherichia coli RRI to ampicillin resistance A part of the cDNA library was used for the conversion. Nitrocellulose colonies inoculate at high density onto the filter and prepare two replica filters for hybridization. (Maniais et al., supra, p. 316). Reduces non-specific background In order to DTA, IMNaCQ, 0.1% NaDodSO-) at 37°C or 42°C. Wash overnight and add 4×5SC (1×SSC=0.15M NaCl2/15mM NaCl2/15mM solution). Sodium enoate), IOX Denhardt solution (IX=0 ．． 02% polyvinylpyrrolidone 10.02% bovine serum albumin 10.02% Ficoll) (Mania Nice, supra, p. 327) at 65°C. Incubate for 3 hours, sonicate, and incubate N. coli DNA at 1000 μg/m (! It was denatured.

Hybridization was performed using 3tp 5°-end labeled oligonucleotides prepared as follows. mixture (6 x l 06 cpm/pmol in lpmol/mQ) - overnight in a solution of the latter. attached to a given oligonucleotide probe. The crossbreeding temperature for each is determined by the empirical formula: tm = 2°C x (dA dT number of bp) + 4°C x (d GdCbp) (where bp means base pair) It corresponded to the temperature Lm. Filters were incubated in JXSSC at hybridization temperature for 1 The cells were washed three times for 30 minutes, dried at room temperature, and subjected to autoradiography. Yang Sexual clones were picked from the master plate and purified through several rounds of screening. did.

The hybridization probes used in the detailed screening procedure were LDL receptor protein Based on sequence information obtained from white matter peptide subfragments, Manufactured.

Purified LDL receptor was digested with CNBr and the internal CNBr fragment was Separated by pressure liquid chromatography (HPLC'), its partial amino acids Sequences were determined by automated Edman degradation. CNBr flag ment to reduced [3H]carboxymethylated receptors (1,6 and 1 ．． Brownlee (B r ownlee) [Santa Clara, California a] Fractionation was performed by reverse phase HPLC using an RP300 column. C listed here NBr peptide was added to the 0.25M Quadrol program or Automated bed using polybrene and non-protein carrier polybrene Two separate separations were performed using a Beckman 890G sequencer. Attached. The yields of NH,-terminal residues of CNBr peptides were as follows for two treatments: They were 400 and 1100 μmol. [3H]Cysteine phenylthiohyl Repetitive yields calculated based on the amount of Dantoin recovered averaged 91%.

All that specify the sequence of amino acids in the two flanking regions of this CNBr fragment synthesized two species of synthetic oligonucleotide probes corresponding to the possible codons of Ta. The oligonucleotide (7) 1-) (7) species designated as A in Table 1 is ( 32 tetradecama coded as Met)-Ala-Glu-Asn-Leu Consists of - The presence of a methionine residue at the amino terminus of this sequence indicates that the peptide This was inferred from the fact that CNBr is generated by CNBr digestion. B and B* in Table 1 A second species of tetradecamer, designated Pro-Glu-(Asp)- Coded 11e-Vat. The Asp residue in this sequence has been assigned twice. This is tentative as this was observed only once during the sequencing process. B/I3” The oligonucleotide species differ in the codon used to specify the Pro residue. (CCT in B.

In B*, 48 members synthesized as subspecies of each 24 members of CCo) It is composed as a whole.

Table 1 CNBr peptide sequence and oligonucleotide synthesis Met Ala Glu Asn Leu Leu Ser Pro Glu (Asp) I le ValCCC TA (, CT A CT ATGGCGA AA T CCGA GA AT GTA G TT A G 　T　A Thirty cross-positive cDNA clones were tested using oligonucleotide species B. Confirmed by screening of NA library. These clones can be subspecies When probed separately with B or B*, 16 clones It hybridized strongly with Tid mixture B, but not with B*. 1 out of 30 clones 2 was positive only for mixture B*. These 28 positive clones were and screened with oligonucleotide mixture A, and both later groups Two plasmids obtained from No. 12 were hybridized with this probe. these two The clone was thought to contain the cDNA for the receptor, so selected for future research.

From two clones crossed with both B* and A oligonucleotide probes. When this plasmid DNA was subjected to restriction endonuclease mapping, results were obtained. The results showed that these two clones were identical. Therefore, these clones This clone was considered to be a representative bovine LDL receptor cDNA clone. pLDL One of these clones, designated R-1, was identified as a true LDL receptor clone. I chose it to make sure it was representative.

To confirm the identity of pLDLR-1, total poly(A)=RNA was isolated from semi-open kidneys and 52P-labeled plasmids were extracted from liver and liver, and nick-translated 52P-labeled plasmids were used as probes. The results were analyzed using a plot test. The RNA plot test was performed as follows. It was. Treating tissues or cells with guanidinium thionanate (mania Nice, supra, p. 196), total RNA was isolated. Poly(A) “RN A was purified by oligo(dT)-cellulose chromatography, and glyoxa 1.5 containing 40mM 3-N-morpholinopropanesulfonic acid. % agarose gel (pH 7,0) for electrophoresis (20 volts, 16 hours). Size fractionation, then 20xS! l; by capillary plotting in C The sample was transferred to a zeta probe membrane (Bio-Rad). spare Crossings and crosses were performed as described in Maniatis supra, p. 320.

When the amount of adrenal RNA is increased, a hybridization signal corresponding to approximately 5.5 kb of mRNA increases. I gradually became stronger. Densitometer scans are obtained for a given amount of adrenal RNA. The signal obtained is nine times stronger than that obtained with the same amount of liver RNA. and showed. Previous studies have shown that functional LDL receptor activity is higher in bovine liver than in bovine liver. of the adrenal glands was about an order of magnitude higher than that in the adrenal glands, but this finding was This is consistent with the difference in the amount of mRNA.

The number of LDL receptors is determined by culturing cells in the presence of cholesterol or related sterols. can be significantly reduced if grown in Poly(A)” RNA, In the absence of sterols (receptor induction) and in the presence of sterols (receptor inhibition) Plot using pLDLR-1 isolated from human A-431 cells grown in Analyzed by. A strong hybridization signal from the approximately 5.5 kb mRNA Detected in NA, this signal was reduced by more than 90% with suppressor RNA.

These results indicate that pLDLR-1 contains at least a portion of the bovine LDL receptor gene. This indicates that it contains cDNA replication. This clone is ATCC #39 It has been deposited as No. 965. Fortunately, the relationship between the Namamugi carrier gene and the human gene is In between, it is possible to use hand sequences as probes to isolate human genes. These procedures are described in Example 2.

Example 2 Cloning of human LDL receptor gene and characteristic materials for human LDL receptor The method for obtaining the full-length cDNA attached is schematically illustrated in FIG. Lawn et al. Cell, 15: 1157-1174 (1978). Screening a human genome library cloned in cteriophages For this purpose, partial bovine cDNA (pLDLR-1) was used. human genome DNA insert Approximately 1×10 bacteriophages containing 32p-labeled pLDLR- 1 was screened. Crossing is done under less severe conditions: 30% holm Aldehyde, 5X SSC, 5X Denhardt's solution, 0.1% SDS (dodecyl sulfur Salmon sperm DNA and lμg/m I2 poly(A), carried out at 42°C. Filter, 2XSSC, 0.1% Washed twice in SDS for 10 min at 22°C and washed once in the same solution for 60 min at 54°C. Washed twice.

A single clone λhl was recognized from this human genome clone library. child This is insert 11k encoding the 3° end of the human LDL receptor gene. b (kilo base pair). 236bp PvuII flag using λhl caused a ment. This fragment is the C0O of human LDL receptor cDNA. It served as a specific probe for the H-terminus.

The cDNA library is available from Okayama and Barb, Molecular & Cell. According to the method of Ra Biology, -;;:-2 l　6　]-170 (1982) It was composed of human fetal adrenal poly(A)'' RNA. was a commercially available enzyme, 2 μg of poly(A)” RNA, 51.5 μg of dT-tailed p cDV1 vector primer and 0.52 pmol dG-tylated linker was used. Following transformation into N. coli HBOI, plasmid c DNA was isolated from approximately 3 x 105 transformants and isolated from Okayama, Barb, Molecule. Cell and Cellular Biology, 3: 280-289 (1983) Longer cDNAs were enriched (6-10 kb) by the sublibrary method.

Human LDL receptor cDNA was transduced with two probes, namely bovine LDL receptor protein. Asn-Glu-Phe-Glu-C with a quality NH at the end A 5°-specific oriform consisting of 32 heptadecamers derived from the ys-Glu sequence. 15 derived from Gonucleotide species and human genome clone λhl (described above). 3”-specific 236 base pair Pvu containing 8bp COOH terminal information sequence (exon) Identification was made using the II fragment. Oligonucleotide is phosporamide It was synthesized by the phosphorai + 1 dite method, and Mark Zoller (Cold Spring Harbor) ・Library) and Ray MacDonald (1”Lay MacDonald ) (Texas Health Science Center (Texas Health 5) Science Center, Dallas). replica filter were screened, and among 2396 recombinants, 5 colonies were 5'- and It was positive for the 3°-specific probe. These clones also have long cDNAs. The plasmid (4.9 kb) with the A insert was named pi01.

The nucleotide sequence of the cDNA insert in the plot is that of LDL receptor mRNA. It was found that the 5° end of the coding region was not included. This cDNA nucleus Otide sequence analysis reveals that the 5°-oligonucleotide probe It was found that there was no hybridization with the region corresponding to the NHt terminus. Rather, pro The NH generated within the coding region was hybridized with an incomplete repeat of the terminal sequence.

Open reading frame pl.

Following the extreme 5' end of the cDNA insert in l, the predicted signal sequence or or there was no evidence of an initiating medionin codon.

To obtain the remainder of the coding region, the sequence near the 5° end of the cDNA in plol was A corresponding oligonucleotide was prepared, and human fetal adrenal poly(A)”R was used as a template. Primer extension using NA was performed. I) Human fetus in BR322 Primer Extended cDNA Live from Child Adrenal Poly(A)” RNA To compose the library, it is complementary to the mRNA strand and the plol cDNA insert. A 20-base synthetic oligonucleotide with 63 nucleotides from the 5° end was used. there was. This library was inserted 8 nucleotides from the 5° end of the plol cDNA insert. Screening was performed using a 20 base second nucleotide resulting in a cleotide. vinegar Among the 1044 recombinants cleaned, one plasmid named p203 The cDNA insert overlaps with plol by 83 nucleotides. and extends almost to the 5° end of human LDL receptor + nRNA. was confirmed.

To construct full-length cDNA, appropriate parts of plol and p203 are ligated. (Figure 2). Due to the lack of convenient restriction enzyme sites, this ligation The procedure requires several partial digestions and the preparation of two intermediate plasmids, as follows: do.

cDNA, which contains the complete translated region of human LDL receptor mRNA, contains duplicated H Joining the p203 insert and the ptot insert via the glAl site. It was constructed by The composition consists of 3 partial digestions and 3 multi-fragment retrievals. , and two intermediate plasmids.

p203 was partially digested with Pstl and then completely with Pvull. A 341 bp fragment was generated. pr, l to HindllT-Pst 1 (518 bp), which is an initial region block of the SV40 virus. Included motor and switching signals (see Figure 2 and Okayama and Barb , 1983). These two fragments were ligated and the PBR322 H Cloned into the indlII-Pvu11 site. This intermediate plasmid was It was named Pl. 394 bp from the 5′ end of pt01 Hg1AI-Ec The oRI fragment was inserted into a 105 bp Pvull-H fragment from the 3' end of p203. giA1 fragment and ligated into EcoR1-PvuII digested pBR322. Gated. This intermediate plasmid was named 1) EPI. 1) EPI, P Partially digested with vuII and then completely with EcoRl to give 499 bp PvuIl-EcoR1 fragment was generated. This DNA7 Ragmen with the 859 bp H4ndllI-PvuII insert from pHPI, and 3° piO corresponding to 85% cDNA and cloning vector The 7 kb H1ndI[I-EcoRl fragment of 1 was ligated.

The final 8.4 kb plasmid, named pLDLR-2, was linked to the SV40 sequence. It contained a full-length cDNA copy of the human LDL receptor (Figure 2).

The result of this genetic manipulation is pLDLR-2, which represents the human LD receptor m All coding regions of RNA (ATCC #39966), all 3-untranslated regions and and a 5,3 kb cDNA integer corresponding to at least a portion of the 5° untranslated region. It was a plasmid containing SERT.

The cDNA insert of pLDLR-2 was derived from Maxam and Gilba. (G11bert), Methods in Enzymology (Meth.

Enzymol, ), 65: 499-500 (+980) and Sanga (S Anger) et al., Proceedings of the National Academy -Of Sciences. Ni Nis A (Proc, Natl, Aca d.Sci.

USA), L±: 5463-5767 (1977). It was. The nucleotide sequence determined for the human LDL receptor cDNA is It is shown in FIG. 3 along with the predicted amino acid sequence of the receptor protein. Shown in Figure 3 5° to the 5° end of the sequence, plasmid pLDLR-2 It contains an unrelated NA that seems to have arisen from the formation of a hairpin loose.

The presence of this extraneous DNA makes this cDNA useful for the diagnostic purposes disclosed by this invention. It does not affect the usefulness of the DNA in any way.

Example 3 Isolation of a genomic recombinant clone corresponding to the normal LDL receptor gene Human LDL receptor A genomic clone spanning the 3° end of the body gene was isolated using bacteriophage vectors. Genomic library constructed in 4A It was separated from The library is located at Harvard University. provided by Mr. Maniatis (T. set The recombinant phage contains 32P-, a cDNA probe for bovine and human LDL receptors. Screened with labeled pLDLR-1 and pLDLR-2 (respectively See Examples 1 and 2). Crossing was carried out under strict conditions: 50% formaldehyde, 5x SSPE (1xssPE=0゜18MNaCf2/10mMNaHtPO4, pH 7, 4/2mM EDTA), 5x Denhardt's solution, o, i% SDS, 100 μg/m (2 salmon sperm DNA and 1 μg/m (! poly(A), 4 The test was carried out at 2°C for 16 hours. The filter is 2xSSC (1xssc=0.15MN aCl210.015M sodium citrate) in 0.1% SDS, 22°C, 1 2 times for 0 minutes and 12 hours at 60'C in 1x SSC and 0.1% SDS. Washed once or more in between. Air dry the washed filter and use Kodak MA R-5 film and DuPont Chronex Lighting Plus Intel (DuPont Cronex Lighting Plus) autoradio at -70°C using a I attached it to the graph.

The 3' end of the normal LDL receptor gene shown in Figure 6 was obtained using the method described above. Clones: λ33-2, λ33-1. It was included on ゛ and λht. Figure 6 The restriction map of the normal LDL receptor gene shown in Note that both existing sequences (or "introns") are also shown. "Ex The term ``son'' refers to the term ``transcribed'' into mRNA and ultimately released into the cell as mature mRNA. It refers to the range of genes that appear in the plasma. One gene has many exons Together, they make up structural genes. Exon is Within genes, they are separated by regions called "intervening sequences" or IVS regions. Ru. The IVS region is transcribed into the first r(NA) transcript of the DNA. However, , unlike exons, ■■S regions are processed outside the scope of initial transcription and are therefore It is not expressed in the ultimate protein product.

), the DNA insert in 33-2 is approximately 12 kb and contains exons A, B and and C code. The insert in λ33-1 is approximately 11 kb and contains an exon. Coded D, E and F. And the insert in λhl is about 11k b, encodes exons E and F. ``P-cDNA shown in Figure 4A The probes include nilobes 2 to 5, which correspond to the following regions of the LDL receptor gene. is the LDL receptor gene shown as approximately 40 kb of DNA in Figure 6. Probe 6 hybridizes with exon in the 5° end of; probe 6 hybridizes with exon C; Lobe 7 hybridizes to the 5' half of exon F; and probes 8 and 9 hybridize to the 5' half of exon F; Crossed with 3° half of Son F. Exon C contains the oxygen-binding sugar region (amino exon D encodes acid residues 693-749); exon D encodes the oxygen-binding sugar region and the membrane spa Eight of the 22 amino acids (residue 7 50-775); exon E encodes the remainder of the membrane-spanning region and the cytoplasm. Encodes 39 of the 50 amino acids (residues 776 to 928) that make up the region; Xon F is the terminal 11 amino acids (residues 829-83) of the cytoplasmic region of the receptor protein. 9) and encodes the 3° untranslated region of mRNA.

Example 4 A case study of a patient with familial hypercholesterolemia. Disclosed in Examples 2 and 3 to characterize mutations in the associated structural genes. This example describes the use of recombinant clones. The indicator case is He is a young male (B, H,) and will be named PH274 hereafter. he is homozygous It has all the clinical features of FH. From PH274 through preliminary functional research The obtained cultured fibroblasts bound +'I-labeled LDL in an amount that was approximately one-third of the normal amount. I found out that it was done. However, the receptor for PH274 is classified as a class in coated pits. and therefore did not transport bound LDL into the cells. That is, PH274 is functionally known as "internalization". zation) defective mutations. Fibers obtained from relatives of P H274 By studying blast cells, he discovered that he had inherited two different mutant alleles. It was. The allele encoding the internalization defective receptor was found in his mother A null (or dormant) allele that is inherited from a parent and does not produce a functional receptor protein It turned out that he had inherited it from his father.

Preliminary biosynthesis studies using cultured fibroblasts of PH274 have shown that internalization is possible. The LDL receptor protein encoded by the defective allele is approximately It was found that the molecular weight was 10,000 Daltons lower. Therefore, this mutation The study began by analyzing genomic DNA obtained from PH274 and his family. I started. As described below, the mutant gene has two exons completely removed. found that the patient had a large deletion that partially removed one exon. .

The deletion mutation discovered in carrying out the present invention for PH274 consists of two repeats D NA element: that is, encodes the membrane-spanning region of the receptor and is responsible for It is thought that this may be the case. Alu sequences from one region of a gene can be isolated from other regions. It may cross-hybridize with other Alu, forming loops in the DNA. follow If this "loop" is removed from the gene, leaving an incomplete gene, the deletion happens.

In the case of PH274, the generated mutant gene has a membrane span region and a cytoplasmic region. produces a truncated LDL receptor that has lost the These truncated receptors Most of them are hidden from the cell, but some of them are bound to the outer surface of the cell. It remains as it is. In this position they can bind LDL, but the cytoplasmic domain These receptors cluster in coated pits and transfer LDL to cells. Preferences of the invention for demonstrating deletion mutations in FH patients A preferred embodiment uses a known technique known as a Southern plot. simple Simply explained, a Southern blot involves first separating a patient's genomic DNA and then This is a method of separating the fragments into fragments and electrophoretically separating them using agarose gel.

The DNA pattern obtained from the gel is then "printed" onto a stable matrix. Ru. The pattern of these fragments corresponding to the LDL receptor gene is then Matrix printed with labeled LDL receptor cDNA or genomic probe By crossbreeding with and visualizing the genetic pattern with markers, we can make everything visible. can be done.

When performing the initial DNA fragmentation, preferably the DNA is fragmented with restriction endonucleotides. Digested by lyase into smaller DNA fragments. however, The only requirement is that the chosen method reproducibly generate the same fragments of genomic DNA. That is, it must be able to be cleaved into patterns. In this way, the equivalent Gene fragments cleaved into fragments are separated by e.g. fragment length. When tested, it shows a reproducible pattern. Therefore, each LDL gene pattern LDL receptor gene fragments from subjects to detect shifts in simply compare it with the corresponding fragment of the control person. The test pattern compared to the control pattern. A shift in the LDL receptor gene fragment in humans is a sign of a mutation in the gene. Probably.

It was determined that this could represent an expressed gene mutation. However, future It is believed that research may require the use of other restriction enzymes. Ru. Therefore, in some cases it may be difficult to find the correct enzyme for a particular deletion. A raw performance test would be useful. Those skilled in the art will recognize the need for such digestive performance testing. You will recognize that you are deaf.

After fragmenting DNA, the generated fragments are then divided into individual fragments. Separate into patterns in which fragments are separated from each other. DNA fragment A preferred method for separation is to electrophorese the DNA in an agarose gel mix. This is to separate the items by size. Agarose gel electrophoresis is known in the art. is a well-known method. But for example column chromatography or density gradient Other separation techniques can also be used, including centrifugation. Gel electrophoresis technology separates The appearance of separated fragments allows accurate determination of size and separation of fragments in a manner that facilitates fragment handling; It is useful in that it allows you to Additionally, LDL receptors present in the gel matrix Gene fragments were directly identified by Southern blotting, which is described in more detail below. You can have a close look.

Figure 4B shows that genomic DNA obtained from normal cells and PH274 was erased with Xbal. A Southern plot is shown after conversion and hybridization with several eDNA probes.

More specifically, Southern plot crosses were performed as follows. Genome DN A (4 μg) was isolated from cultures of fibroblasts from the indicated subjects. Nice, above), Xba I (-New England Biolabs) England Biolabs)), buffer A (40mM) lysacetate salt, 3mM NatEDTA, 20o+MNaOAc, and 18mM NaC ( ! Electrophoresis was performed in 1% agarose containing 1% chloride, pH 8, 15), and osmotic diffusion was performed. and transferred to nitrocellulose paper (Mania Nice, supra). paper, 50% holm Amide, 1% SDS, 5x Denhardt's solution, 5x SSPE and 100 μg/m Q 3 in Elysia coli DNA! 1 at 42°C without P-labeled probe. After precrossing for an hour, the appropriate 5tp-labeled cDNA probe (2 Cultured at 42°C with ~4 x 10' cpm/m. After crossbreeding, paper, 1% Washed with SDS and 2X SSC for 15 min at 23°C, then 1% SDS and 0.1X5SC (for probe l hybridization) or 1% SDS and 0. Washed in 5SSC (for probes 2-9) for 4 hours at 68°C. filter was exposed to intensified screened X-ray film at -70°C for 24 hours. probe A representative plot cross using 1 and 8 is shown in Figure 4B.

31p-labeled cDNA probe used to map the LDL receptor gene. Size and location are indicated by filled bars in Figure 4A and numbered 1-9. . Probe 1 (double-stranded DNA) is a 2.1 kb EcoRI from piO1 -Sμ fragment and three different 0, 9 kb BamHI-XhoI fragments. a mixture of fragments that together cover most of the translated region of a gene. spanned. Fragments were analyzed by polyacrylamide gel electrophoresis and electrolysis. Purified by separation, then by fine barb (Peinberg) and four V ogelstein, Analytical Biochemist Lee (Anal, Biochem, ), ±32: 6-13 (1983) Labeled with 3fp after random hexanucleotide priming as described in did. Probes 2-9 are Church and Gilbert 1 bert), Proceedings of the National Academy - Fist of Science, N.N.A., 81: 1911-1995 (1984). The method uniformly 31p-labeled the M13 subclone of pLDLR-2. The DNA was prepared as a single-stranded DNA with a length of approximately 100 nucleotides. All probes are , had a characteristic radioactivity of at least 5 x 10 scpm/μg.

Briefly, in order to prepare probe 2, meshing, Sods in Enzymology, No. 101: 20-78 (1983). A DNA fragment containing nucleotides 267 to 1080 (Figure 3) as shown. The fragment was cloned into the bacteriophage M13mp9 vector. evenly A 32P-labeled single-stranded DNA probe approximately 100 bases in length was prepared by Church et al. M+3 universal primer from clones obtained by the method of J. and Gilbert, supra. , and three unlabeled deoxynucleotides and one alpha 32p - DNA polynucleotide for primer extension in the presence of labeled deoxynucleotides It was prepared using the Klenow fragment of merase I. profit The radioactive primer extender was boiled and placed on a denaturing acrylamide gel. denatured from the template by size fractionation, electroelution, and then directly plated. Used as an immer. Probes 3 to 9 use different regions of the sequence shown in Figure 3 as templates. It was prepared using the same procedure except for using the M13 clone containing .

Referring to FIG. 3, probe 3 has nucleotide numbers 719 to 2. 544, probe 4 contains nucleotide numbers 267-1078, Probe 5 contains nucleotide numbers 1573-3486, and probe 6 contains nucleotide numbers 1573-3486. , contains nucleotide numbers 2154 to 2544, and probe 7 contains nucleotide numbers 2154 to 2544. Containing nucleotide numbers 2545-3948, probes 8 and 9 are Genome DN from normal cells and PH274 after code numbers 4508-496 A Southern plot of A is shown. Probe 1 is the translated region of LDL receptor mRNA It is a mixture of mostly spanned cDNAs (Fig. 4A). This probe successfully When crossed with Xbal-digested genomic DNA from humans, the results are shown in A, C, and D. Three bands are observed at 23 kb, 10 kb and 7 kb, respectively (Fig. 4B). ). All of these normal bands and an additional 13 kb band designated B are PH274 present in the genomic DNA of These findings suggest that the mutant allele of PH274 one has a normal restriction pattern, whereas the other allele gives rise to band B. It suggests that.

In order to localize the DNA segment responsible for the generation of band B, the XbaI digest was probed with a short DNA fragment corresponding to the L receptor cDNA (probe 2-9, Figure 4A). Probes 2-5 are used in normal and PH274 DNA. They interbred and became the same band. Probes 6.8 and 9 (excluding probe 7) ) were hybridized to form an unusual 13kb band in PH274. Band B is probe 7 probes on either side of probe 7 (i.e. probe 6.8 and 9), this fragment is included in probe 7. This is thought to result from a deletion in the DNA that includes the mRNA-encoding region. It will be done. This deletion was created by fusing adjacent DNA fragments to create a single 13kb fragment. The removal of at least one Xba1 site with the occurrence of band B It probably includes a lot of things.

Unique mutations expressed by PH274 are also found in other FH patients It is never to be interpreted as the only type of mutation.

Therefore, Probes 2 to 5 are the altered LDL receptor gene profiles for PH274. Although unable to show a fragmentation pattern, these probes were similar to other FH It may be useful to detect other mutations in patients. Probes 2 to 5 are Cross with 5° half of the body gene. Therefore, the occurrence of this region of the receptor gene Mutations will be detected using probes 2-5. Similarly, probe 7 crosses with Obi (Fig. 4A), so mutations occurring in this gene region can be detected. would be useful.

Band B is caused by the maternal (internalization defective) allele or the paternal (absent) allele. Genomic DNA obtained from two parents to determine which of the two parents it originated from bal digestion was performed. Figure 5 shows that band B exists in the mother's DNA, but the father's This indicates that the deletion is not an internalization defect. The presence of a mutation in the LDL receptor gene was detected, indicating that it was present in the defective allele. can be used to specifically examine such mutations in detail. The following shows that the present invention is useful in providing a method capable of identifying Shown by experiment.

Southern PCR using genomic recombinant clones and LDL receptor cDNA probes The lot method can be adopted to create a detailed restriction map of PH patients. these Genetic mapping techniques are now well known to those skilled in the art. PH27 A specific map made for 4 is shown in Figure 6. Figure 6 shows the normal LDL receptor gene. The restriction maps of the 3° end of the gene and the gene carrying the deletion from PH274 are compared.

The lower scale indicates the length of the genomic DNA in kilobases. Normal LDL reception The composition of the patient genes is shown at the top of the figure. Exons are filled-in segments. is represented by an asterisk and an uppercase letter. Intervening sequences (IVS) are blank segments. represented by uppercase and lowercase letters. Alu repeats in IVSc and exon F The array is shown. Restrictions used to define gene deletions in PH274 Enzyme recognition sites are shown.

The restriction map of the normal receptor gene consists of three genomic clones (λ33-2, λ33- 1. It was created from the research of λhl). The gene map in PH274 is Made from λFH274-10 containing fragment membranes (Figures 4B and 5) (See below). Filled bars above or below the restriction map indicate DNA sequencing. Represents the normal and mutant gene segments used.

Genomic DNA5 from fibroblasts of PH274 to obtain λF)1274-10. 70 μg was prepared and digested with Xba11700 units. The digested DNA is Extracted with phenol/chloroform, then chloroform and 70% ethane. Buffer B (10 m M Tris-HCl and 1mM Na*EDTA, pH 7.5). D.N. A (80 μg) was re-digested with XballOO units and diluted with 1% low agarose gel (Bethesda Research Laboratories) In addition to 40V, 4 Electrophoresis was performed at ℃ for 72 hours. After electrophoresis, DNA fragments ranging from 9 to 23 kb were Extract 10 2 mm mm slices of gel containing fragments, concentrate, and buffer B2. Dissolved in 0μQ. A small amount (4 μm) of each DNA fraction, F) (X clause from 274 5 μg of digested genomic DNA and size marker fragments in buffer A. Add to individual lanes of a 0.8% agarose gel and incubate for 16 hours at 35V and 23°C. It was subjected to pneumophoresis.

DNA was transferred to nitrocellulose paper and treated with 5ap-labeled probe as described above. It was crossed with Bull. The obtained autoradiogram shows an abnormal 13kbXba! Hula Both fragments containing the fragment (Fragment B, Figure 4) were recognized. This fraction (10 The remaining DNA from the 500n T4 DNA ligase (Ninny England Biolabs) 49 g 0 units at 14° C. for 72 hours. ligated substances in vitro. , encased in lambda phage particles (Amersham) Amount of 6,7×1O” plaque-forming units was obtained. This library was used as a 31p- Screening was performed using labeled clone 8 (Figure 1). This probe 8 is compatible with The normal fragment (7kb) is too small to generate visible recombinant phages. Therefore, it was expected that only abnormal fragments would be detected. one recombinant clone The pattern was confirmed (λFl(274-10)) and one cycle of plaque purification was performed. separated after being separated. A 13 kb insert into λPI (274-10) was added to the purified DN psP65 (Promega Biot ea)) and used for mapping of restriction endonucleases. .

Figure 6 shows that the Pvu11 site in IVSc and the 5st1 site in the exon are The cloned fragments of the normal gene are separated by about 5 to 8 kb, but the PH The cloned fragments of the 274 genes are separated by approximately 0.6 kbL. It shows that there is no. Furthermore, several species between the Pvu11 site and the 5st1 site The restriction enzyme site was missing from the cloned PH274 gene. It is shown. These data were analyzed using the genomic Southern plot described above. This test confirms the deletion of 5kb of DNA from the PH274 gene. This suggests that he did.

The deletion separated the 3′ end of IVSc, all of exons D and E, and IVS, and the 5° end of exon F.

The location of the 5′ and 3° break points and the structure of the deletion bond in PH274 were precisely determined. To identify the nucleotides of the cloned portion of the gene delimited by the bars in Figure 6. The sequence was determined. From these sequences, the deletion linkage indicates that the oppositely oriented Δb species It turns out that this is happening between two repeating elements. The 5° side of the deletion bond is IVS was derived from the Alu sequence in e. The 3° side of the deletion junction is located oppositely in exon F. was derived from the Alu sequence.

A second PH patient was tested using the present invention, and this patient's LDL No deletion of the condition gene could be detected. This worker's LDL receptor genetics The deletion in the offspring is likely due to a point mutation. Caused by deletion mutation Defects that are not caused by the I can do it.

Although recombination between repetitive DNA sequences is hypothesized to be responsible for deletions, our According to knowledge, such rearrangements have not been reported in eukaryotic cells. most Well-characterized mammalian deletion mutations, namely human alpha and beta -In mutations that occur in the globin gene, one of the deletion points is located in the wholesale sequence. Other breakpoints, although frequent, have always occurred in unique sequences of DNA. was.

The present invention provides examples of what the inventors believe to be a preferred method for carrying out the invention. I have explained the light. However, they are by no means the only modes of carrying out the invention. I don't mean that. For example, the restriction enzyme XbaI has PH274. Although useful in demonstrating one specific mutation, certain sudden changes in other PH patients Other restriction enzymes may be required to indicate mutations. Similarly, we found that Southern ・The plotting method is the best method for representing the pattern of LDL receptor gene fragments. However, other methods may be employed and are considered to be within the scope of the invention. should be For example, fragments can be separated by column chromatography. However, it is also possible to analyze the presence of gene fragments as they elute from the column. It is possible. Similarly, the probes used in practicing the present invention are radiolabeled. However, radiolabeling was considered the only way to make the probe detectable. Must not be allowed. For example, DNA hybridization probes can be labeled with lightning isotopes. Alternatively, it can be labeled by binding a ligand such as biotin. Good too. In general, any ligand capable of specifically binding that can be detected independently can be used. can be used. These and any other modifications are included within the scope of the following claims. You have to think that it will happen.

Although the specific case disclosed is an abrupt change resulting from a deletion, it is possible to extend the invention further. sudden changes resulting from other types of causes, including single nucleotide changes. Mutations can also be detected. The latter results in loss or gain of restriction enzyme cleavage sites. If it does, it can be directly detected using a Southern gel plot. single nucleus Otide changes also occur in chemically synthesized oligonucleotides that correspond to the cDNA sequence. It can be detected by reductive hybridization with short segments of cDNA. weakened Hybridization occurs after the mutant and normal DNA sequences have crossed with each other and are then "melted". Alternatively, it can be detected by dissociation. Such abnormal melting behavior , heat, low ionic strength, or hybridized DNA strands such as urea and formamide. Detected as increased sensitivity to dissociating drugs. Therefore, the present invention provides LD It is useful in diagnosing all possible mutations of the L receptor gene.

Igo-・1ll-＾p--1店t+,, PcT/IJS 85102461AN NEX TQ τF, E INTERNATIONAL 5EARCF, REP ORT　NINTERNATIONAL　APPLICAτIoN　No,　 PCT/US 8510246m (SA 11738)

Claims (1)

[Claims] 1. At least the second DNA sequence encoding the human LDL receptor protein can be hybridized. A recombinant DNA transfer vector comprising a first DNA sequence complementary to a portion of -. 2. The first DNA sequence is at least a hybridizable portion of human LDL receptor mRNA. of claim 1 further defined as a cDNA sequence complementary to Recombinant DNA transfer vector. 3. The first DNA sequence is replaced as at least a hybridizable portion of the DNA sequence of FIG. A recombinant DNA transfer vector according to claim 1 as defined in claim 1. 4. The first DNA sequence is replaced as at least a hybridizable portion of the DNA sequence of FIG. The recombinant DNA transfer vector according to claim 2, defined in . 5. Recombinant DNA according to claim 1, further defined as a recombinant plasmid. A transfer vector. 6. The composition of claim 1 further defined as a recombinant bacteriophage. Replacement DNA transfer vector. 7. The set of claim 5 further defined as plasmid pLDLR-2. Replacement DNA transfer vector. 8. The bacteriophage is further defined as bacteriophage λ33-1. The recombinant DNA transfer vector according to claim 6. 9. The bacteriophage is further defined as bacteriophage λ33-2. The recombinant DNA transfer vector according to claim 5. 10. The bacteriophage is further defined as bacteriophage λhI. The recombinant DNA transfer vector according to item 5. 11. At least a hybridizable second DNA sequence encoding human LDL receptor protein A recombinant DNA transfer vector comprising a first DNA sequence that is coffin-like to the functional part. Bacterial strains with tar. 12. the first DNA sequence is at least a hybridizable portion of human LDL receptor mRNA; Claim 1 further defined as a cDNA sequence complementary to recombinant DNA transfer vector. 13. The first DNA sequence is at least a hybridizable portion of the DNA sequence in FIG. A bacterial strain according to claim 11 as further defined. 14. Claim 11, wherein the bacterial strain is identified as ATCC #39966 bacterial strains. 15. Further defined as having a DNA sequence encoding the amino acid sequence shown in Figure 3. The recombinant DNA transfer vector according to claim 1, wherein the recombinant DNA transfer vector is 16. At least a hybridizable second DNA sequence encoding human LDL receptor protein A DNA sequence comprising a first DNA sequence that is complementary to a functional moiety. 17. the first DNA sequence is at least a hybridizable portion of human LDL receptor mRNA; Claim 16 further defined as a cDNA sequence complementary to The DNA sequence of 18. The first DNA sequence is at least a hybridizable portion of the DNA sequence in FIG. A DNA sequence according to claim 16 as further defined. 19. The first DNA sequence is at least a hybridizable portion of the DNA sequence in FIG. A DNA sequence according to claim 17 as further defined. 20. The first DNA sequence consists of probe numbers 1, 2, 3, and 4 in Figure 4A. 23. The method of claim 22, defined as: 25. Separation of DNA fragments using a gel matrix 23. The method of claim 22, further defined as subjecting to electrophoresis. 26. If the gel matrix is an agarose gel matrix or polyacrylic 26. The method of claim 25 further defined as a midgel matrix. 27. Separation of DNA fragments is performed by chromatography of DNA fragments. 23. The method of claim 22 further defined as subjecting to separation. 28. Separation of DNA fragments involves subjecting the DNA fragments to velocity sedimentation. 23. The method of claim 22, further defined as: 29. Claim 1, wherein the preselected restriction endonuclease is XbaI. 22. The method described in 22. 30. Confirmation of the pattern of DNA fragments corresponding to the LDL receptor gene The labeled LDL receptor gene DNA fragment is added to step (b) 5, 6, 7, 8 or 17. The DNA sequence according to claim 16, which is 21. If the first DNA sequence is probe number 1, 6, 8 or 9 in Figure 4A, The DNA sequence according to item 16. 22. A method for diagnosing a mutation in a human LDL receptor gene, the method comprising: (a) a human LDL receptor gene mutation; Divide the DNA from the cells and (b) separate the DNA fragments from step (a) into their (c) corresponds to the LDL receptor gene. (d) normal LDL receptor gene D Confirm changes in the pattern of step (c) by comparing with the pattern of the NA fragment. A method consisting of diagnosing a mutation by identifying 23. DNA fragmentation is performed by pre-selected restriction endonucleases. 23. The method of claim 22, further defined as digesting. 24. The physicochemical properties are separated from at least one of molecular weight or total charge. the pattern of the hybridized fragment using a label. 23. The method of claim 22, further defined as confirming a turn. 31. The label is a radiolabel and autoradiography detects hybrid fragments. 31. The method according to claim 30, for confirming a pattern of marks. 32. The DNA fragment is a second DNA encoding human LDL receptor protein. at least one of the first DNA sequences that is complementary to at least a hybridizable portion of the sequence; 31. The method of claim 30, further comprising a hybridizable portion. 33. The DNA fragment is a cDNA insert of plasmid pLDLR-2. The method according to claim 30, comprising a hybridizable portion of DNA derived from Law. 34. The DNA fragment is at least hybridizable with human LDL receptor mRNA. A claim comprising at least a hybridizable portion of a cDNA that is complementary to a portion The method according to item 30. 35. The DNA fragment includes at least a hybridizable portion of the DNA sequence in Figure 3. 31. The method of claim 30, comprising: 36. The DNA fragment is at least hybridizable with plasmid pLDLR-2. 31. The method of claim 30, comprising: 37. The DNA fragment is at least hybridized with bacteriophage λ33-1. 31. The method of claim 30, comprising a possible portion. 38. The DNA fragment is at least hybridized with bacteriophage λ33-2. 31. The method of claim 30, comprising a possible portion. 39. The DNA fragment is at least hybridizable to bacteriophage λh1. 31. The method of claim 30, comprising: 40. The DNA fragments are probe numbers 1, 2, 3, 4, 5, and 6 in Figure 4A. , 7, 8 or 9. 41. If the DNA fragment is probe number 1, 6, 8 or 9 in Figure 4A. 31. The method according to claim 30.